CN102181384A - Acinetobacter calcoaceticus T32 capable of metabolizing furazolidone and application thereof - Google Patents
Acinetobacter calcoaceticus T32 capable of metabolizing furazolidone and application thereof Download PDFInfo
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- CN102181384A CN102181384A CN 201110049039 CN201110049039A CN102181384A CN 102181384 A CN102181384 A CN 102181384A CN 201110049039 CN201110049039 CN 201110049039 CN 201110049039 A CN201110049039 A CN 201110049039A CN 102181384 A CN102181384 A CN 102181384A
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- nifurazolidone
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Abstract
The invention relates to the field of treating environment by using microorganisms, in particular to acinetobacter calcoaceticus T32 capable of metabolizing furazolidone, and application thereof. The acinetobacter calcoaceticus T32 has been registered and collected in the China General Microbiological Culture Collection (CGMCC) on sixth, November, 2010; and the collection number is CGMCC No.4311. The acinetobacter calcoaceticus T32 can be used as bactericide for degrading the furazolidone. The furazolidone in the environment is removed by the metabolic process of T32, and the acinetobacter calcoaceticus T32 has the advantages of high degradation speed, low cost, convenience of operation and the like.
Description
Technical field
The present invention relates to utilize microorganism to carry out the environmental improvement field, but the acinetobacter calcoaceticus of metabolism Nifurazolidone and application thereof specifically.
Background technology
Nifurazolidone has another name called Trichofuron, and molecular formula is C
8H
7N
3O
5, be a kind of extensive pedigree antibiotic of synthetic with 5-nitrofuran basic structure.Because Nifurazolidone has advantages such as low price, drug effect be fast, be commonly used to prevent and control cholera, coccidiosis, swine enteritis and turkey blackhead disease etc. because of the infection that bacterium and protista cause, certain application is also arranged in aquaculture.Yet studies show that Nifurazolidone can make the sudden change of laboratory animal producer and bring out canceration.The residual chronic poisoning that cause fish and shrimp of Nifurazolidone in culture environment of aquatic products seriously jeopardizes healthy fish and cultures.Classify Nifurazolidone as forbidden drug in the world at present, also the detecting be limited to and detect of Nifurazolidone in the regulation animal derived food sent out in Ministry of Agriculture's file agriculture and animal husbandry of China [2002] No. 1, and issued the ban that bans use of such veterinary drug.At present, compare with monitoring with respect to the detection of Nifurazolidone, research report how to remove the Nifurazolidone in environment and the organism is less.Compare physicochemical Nifurazolidone removing method, biological treatment has thorough, the advantages such as expense is low, non-secondary pollution of degraded, add that microorganism itself has and stronger has that accommodative ability of environment is strong, the metabolism diversity advantages of higher, multiple organic pollutant in the environment is had metabolism, can reduce the toxicity of pollutent or the pollutent in the removing environment by himself metabolism.At present, the research of related microorganism metabolism Nifurazolidone only has Jiang Lijuan etc. to screen the Pseudomonas aeruginosa of a strain degradable lower concentration Nifurazolidone from the bed mud of contaminated fish pond, removes for utilizing bacterium that Nifurazolidone pollutes the data that provides the foundation in the environment.
Summary of the invention
But the object of the invention is to provide a kind of acinetobacter calcoaceticus and application thereof of metabolism Nifurazolidone.
For achieving the above object, the technical solution used in the present invention is: (behind the final version content in claims being copied to herein)
Compared with prior art, positively effect of the present invention is:
At present, be that the environmental pollutant clearance technique of key link all has application in the degraded of heavy metal accumulation, organic contamination and oil degradation with the bacterial metabolism approach.Nifurazolidone in the acinetobacter calcoaceticus degradable environment that the present invention obtains has advantages such as degradation speed is fast, expense is low, operation steps is simple, for the furazolidone residual of thoroughly removing in the environment provides thinking preferably.
Description of drawings
The sequence of the 16S rDNA of the acinetobacter calcoaceticus T32 that Fig. 1 provides for the embodiment of the invention.
In the acinetobacter calcoaceticus T32 degraded Nifurazolidone process that Fig. 2 provides for the embodiment of the invention, increase the relative concentration change procedure of Nifurazolidone in the solution in time.The initial concentration of Nifurazolidone is 5ppm; X-coordinate is a fate, and ordinate zou is the relative concentration of Nifurazolidone.(■, the relative concentration of Nifurazolidone in the abacterial solution; △ contains the relative concentration of Nifurazolidone in the solution of bacterium T32).
Fig. 3 is in the acinetobacter calcoaceticus T32 degraded Nifurazolidone overshoot of the embodiment of the invention, increases the relative concentration change procedure of Nifurazolidone in the solution in time.The initial concentration of Nifurazolidone is 10ppm; X-coordinate is a fate, and ordinate zou is the relative concentration of Nifurazolidone.(■, the relative concentration of Nifurazolidone in the abacterial solution; △ contains the relative concentration of Nifurazolidone in the solution of bacterium T32).
Fig. 4 is provided for the acinetobacter calcoaceticus CGMCC No.4311 that utilizes the embodiment of the invention and provide by 30 hours the HPLC spectrogram of Nifurazolidone of 5ppm.
Embodiment
Acinetobacter calcoaceticus T32 16S rDNA obtains:
Described acinetobacter calcoaceticus T32 is to register preservation in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC); Preservation date is on November 06th, 2010; Be numbered CGMCC No.4311.The acinetobacter calcoaceticus T32 that collection is in stationary phase extracts the genomic dna of this bacterium.Concrete steps are:
The T32 bacterial strain of (1) 30 ℃ of incubated overnight is inoculated in the test tube that contains the 10mlLB substratum by the inoculum size of 1% (V/V), treats the OD of bacterium
600During ≈ 1.0 left and right sides, get 1ml bacterium liquid in the 1.5ml centrifuge tube 10, the centrifugal 1min of 000g collects thalline.
(2) add 1ml 0.9%NaCl washing precipitation, 10, the centrifugal 3min of 000g.
(3) abandon supernatant, with the 450 μ l TE damping fluids precipitation that suspends again, and add the SDS of 50 μ l 20%, the centrifuge tube that turns upside down gently makes it mixing, places 5min in 75 ℃ of water-baths, becomes clarification to the cracking of bacterium liquid; Described TE damping fluid is 12mM Tris-HCl, 12mM EDTA, and pH 8.0.
(4) add 3: 1 phenol of 500 μ l volume ratios-chloroform extracting, put upside down gently and make it mixing, room temperature leaves standstill behind the 5min 10, the centrifugal 5min of 000g.
(5) supernatant is transferred to a new 1.5ml centrifuge tube and supply volume to 500 μ l, adds phenol-chloroform of 3: 1 of 500 μ l volume ratios, the mixing room temperature leaves standstill behind the 5min 10, the centrifugal 5min of 000g.Again supernatant is transferred in the new centrifuge tube, so repeatedly, till can't see the intermediary protein layer.
(6) supernatant is transferred to a new 1.5ml centrifuge tube and supplies volume to 500 μ l, adds 500 μ l chloroforms, gently behind the mixing 10, and the centrifugal 5min of 000g.
(7) supernatant is transferred to a new 1.5ml centrifuge tube, adds TE damping fluid to 500 μ l.The 3M NaAc mixing that adds 50 μ l then adds the Virahol of 550 μ l at last, turns upside down several times, should see having flocks to produce this moment, and 10, the centrifugal 5min of 000g.
(8) abandon supernatant, add the washing with alcohol precipitation of 1ml 70%, 10, the centrifugal 5min of 000g blots liquid as far as possible, the about 10min of drying at room temperature.
(9) add 50 μ l and contain the TE damping fluid dissolving DNA that final concentration is 10-20mg/ml RNaseA.Utilizing 8F (AGAGTTTGATCCTGGCTCAG) and 1492R (GGTTACCTTGTTACG ACTT) is primer, and the T32 genomic dna of extraction is a template, and its 16S rDNA of pcr amplification also identifies.The condition of PCR is 94 ℃ of sex change 4min, 94 ℃ of sex change 30s, and 57 ℃ of annealing 1min, 72 ℃ are extended 1min, since second step, 30 circulations.The PCR product is checked order by Beijing promise match gene.Dna sequencing fragment (referring to Fig. 1).
Utilize acinetobacter calcoaceticus T32 to handle and contain the method for concentration for the Nifurazolidone solution of 5ppm:
(1) microorganism strains: select acinetobacter calcoaceticus T32 CGMCC No.4311 for use.
(2) seed culture:: choose a ring bacterium colony from flat board and insert the LB substratum, shake bottled liquid measure 30%, culture temperature is 28～30 ℃, cultivates rotating speed 180～200rpm/min, and incubation time is 10-16h.The prescription of LB substratum is: Tryptones, 10g, yeast extract, 5g, sodium-chlor, 10g, distilled water, 1L.
(3) domestication is cultivated: the T32 in the step (2) is seeded in the inorganic salt protein culture medium 28 ℃ of incubated overnight 10-16h by the inoculum size of 1% (V/V).The prescription of inorganic salt protein culture medium is: Na
2HPO
412H
2O 3g/L, KH
2PO
41g/L, NaCl 3g/L, MgSO
40.3g/L, Tryptone 2.5g/L; Regulate pH6.0-8.0.
(4) T32 degraded Nifurazolidone: is 1 * 10 with the T32 thalline in the step (3) by initial concentration
8CFU mL
-1Be inoculated in the inorganic salt peptone liquid nutrient medium that contains the 5ppm Nifurazolidone and cultivate, in contrast with the inorganic salt peptone liquid nutrient medium that contains Nifurazolidone of not inoculating T32.Take out the nutrient solution of 500 μ l at different time points, 13, the centrifugal 5min of 000rpm collects supernatant liquor.
(5) high performance liquid chromatography of Nifurazolidone concentration (HPLC) detects: the supernatant liquor of collecting in the step (4) is carried out high performance liquid chromatography respectively detect, measure the relative concentration of Nifurazolidone, measure 30 hours chromatograms of Nifurazolidone of handling 5ppm simultaneously.The high performance liquid chromatography testing conditions is: liquid chromatograph: skyray LC-310 liquid chromatographic system; Chromatographic column: the reverse post 4.6 * 250mm of Waters C18, granularity 5 μ m; Testing conditions is a moving phase, V (acetonitrile): V (acetic acid aqueous solution) [V (acetate): V (water)=1: 1000]=55: 45; Sample size is 20 μ L; Temperature is 25 ℃; The UV-detector detect parameters is for detecting wavelength 365nm.
Starting point concentration with the adding Nifurazolidone is 100%, the relative concentration of Nifurazolidone in the different time points solution=(the starting point concentration peak area of the peak area/Nifurazolidone of sample) * 100% is increased the curve (referring to Fig. 2) of Nifurazolidone relative concentration in the solution in view of the above in time.Starting point concentration with the adding Nifurazolidone is 100%, and under the situation that does not add bacterium, the concentration of Nifurazolidone becomes 90% from 100% in the time of 6 days; And adding acinetobacter calcoaceticus T32, the concentration of Nifurazolidone was reduced to 0.9% when the concentration of Nifurazolidone was reduced to 7.2%, 6 day from 100% when cultivating 1 day.As seen, be in the Nifurazolidone solution of 5ppm at starting point concentration, add bacterium T32, can be with about 90% Nifurazolidone degraded in the solution.
Acinetobacter calcoaceticus CGMCC No.4311 handles 30 hours the HPLC spectrogram (referring to Fig. 4) of Nifurazolidone of 5ppm simultaneously.The Nifurazolidone peak area that does not add bacterium as can be known is far longer than and adds bacterium Nifurazolidone peak area later on.
Utilize acinetobacter calcoaceticus T32 to handle and contain the method for concentration for the Nifurazolidone solution of 10ppm:
(1) microorganism strains: select acinetobacter calcoaceticus T32 CGMCC No.4311 for use;
(2) seed culture: from-80 ℃ of refrigerators the T32 that preserves is connected in the LB substratum of 10ml, shakes bottled liquid measure 30%, culture temperature is 28～30 ℃, cultivates rotating speed 180～200rpm/min, and incubation time is 10-16h.The prescription of LB substratum is: Tryptones, 10g, yeast extract, 5g, sodium-chlor, 10g, distilled water, 1L.
(3) domestication is cultivated: the T32 in the step (2) is seeded in the inorganic salt protein culture medium 28 ℃ of incubated overnight 10-16h by 1% inoculum size (V/V).The prescription of inorganic salt protein culture medium is: Na
2HPO
412H
2O 3g/L, KH
2PO
41g/L, NaCl 3g/L, MgSO
40.3g/L, Tryptone 2.5g/L; Regulate pH6.0-8.0.
(4) T32 degraded Nifurazolidone: is 1 * 10 with the T32 thalline in the step (3) by initial concentration
8CFU mL
-1Be inoculated in the inorganic salt peptone liquid nutrient medium that contains the 10ppm Nifurazolidone and cultivate, in contrast with the inorganic salt peptone liquid nutrient medium that contains Nifurazolidone of not inoculating T32.Take out the nutrient solution of 500 μ l at different time points, 13, the centrifugal 5min of 000rpm collects supernatant liquor.
(5) calculating of the high performance liquid chromatography of Nifurazolidone concentration (HPLC) detection and degradation rate: the supernatant liquor of collecting in the step (4) is carried out high performance liquid chromatography respectively detect, measure the relative concentration of Nifurazolidone, the high performance liquid chromatography testing conditions is: liquid chromatograph: s kyray LC-310 liquid chromatographic system; Chromatographic column: the reverse post 4.6 * 250mm of Waters C18, granularity 5 μ m; Testing conditions is a moving phase, V (acetonitrile): V (acetic acid aqueous solution) [V (acetate): V (water)=1: 1000]=55: 45; Sample size is 20 μ L; Temperature is 25 ℃; The UV-detector detect parameters is for detecting wavelength 365nm.Starting point concentration with the adding Nifurazolidone is 100%, the relative concentration of Nifurazolidone in the different time points solution=(the starting point concentration peak area of the peak area/Nifurazolidone of sample) * 100% is increased the curve (referring to Fig. 3) of Nifurazolidone relative concentration in the solution in view of the above in time.Under the situation that does not add bacterium, the concentration of Nifurazolidone is almost without any variation in the time of 6 days; And adding acinetobacter calcoaceticus T32, the concentration of Nifurazolidone was reduced to 10% when the concentration of Nifurazolidone was reduced to 27%, 6 day from 100% when cultivating 1 day.As seen, be in the Nifurazolidone solution of 10ppm at starting point concentration, add bacterium T32 and cultivated 6 days, can be with the degraded of the Nifurazolidone more than 90% in the solution.
Claims (5)
1. but the acinetobacter calcoaceticus of a metabolism Nifurazolidone is characterized in that: acinetobacter calcoaceticus Acinetobacter calcoaceticus T32, in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) registration preservation; Preservation date is on November 06th, 2010; Be numbered CGMCC No.4311.
2. but the application of the acinetobacter calcoaceticus of the described metabolism Nifurazolidone of claim 1 is characterized in that: described acinetobacter calcoaceticus can be used as the microbial inoculum of degraded Nifurazolidone.
3. but press the application of the acinetobacter calcoaceticus of the described metabolism Nifurazolidone of claim 2, it is characterized in that: described acinetobacter calcoaceticus degraded Nifurazolidone process is: be incubated in the inorganic salt peptone liquid nutrient medium that contains Nifurazolidone after the acinetobacter calcoaceticus domestication is cultivated and cultivate, utilize high performance liquid chromatography (HPLC) to detect its degradation rate, in contrast with the inorganic salt peptone liquid nutrient medium that contains Nifurazolidone of not inoculating T32.
4. but by the application of the acinetobacter calcoaceticus of the described metabolism Nifurazolidone of claim 2, it is characterized in that: inorganic salt peptone liquid nutrient medium is Na in the described inorganic salt peptone liquid nutrient medium that contains Nifurazolidone
2HPO
412H
2O 3g/L, KH
2PO
41g/L, NaCl 3g/L, MgSO
40.3g/L, Tryptone 2.5g/L; Regulate pH to 6.0-8.0.
5. but press the application of the acinetobacter calcoaceticus of the described metabolism Nifurazolidone of claim 2, it is characterized in that: described high performance liquid chromatography (HPLC) testing process of utilizing is for adopting liquid chromatograph: skyray LC-310 liquid chromatographic system; Chromatographic column: the reverse post 4.6 * 250mm of Waters C18, granularity 5 μ m; Testing conditions is a moving phase, V (acetonitrile): V (acetic acid aqueous solution) [V (acetate): V (water)=1: 1000]=55: 45; Sample size is 20 μ L; Temperature is 25 ℃; The UV-detector detect parameters is for detecting wavelength 365nm.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102676418A (en) * | 2011-10-28 | 2012-09-19 | 中国科学院烟台海岸带研究所 | Application of acinetobacter calcoaceticus capable of degrading petroleum |
| CN102851236A (en) * | 2012-08-01 | 2013-01-02 | 上海交通大学 | Acinetobacter and construction method and application thereof |
| CN103013851A (en) * | 2012-10-08 | 2013-04-03 | 上海交通大学 | Complex microbial inoculant and applications thereof |
| CN106754473A (en) * | 2016-11-24 | 2017-05-31 | 中国科学院烟台海岸带研究所 | One planting sand good fortune bacillus and the application in open maritime environment pollution thing is repaired |
| CN107058156A (en) * | 2016-12-27 | 2017-08-18 | 北京市农林科学院 | Tetracycline antibiotics degradation bacteria strains, the microbial inoculum containing the bacterial strain and its application |
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| CN101899404A (en) * | 2010-01-05 | 2010-12-01 | 广东省微生物研究所 | Acinetobacter calcoaceticus and application thereof in degrading decabromodiphenyl ether |
| CN101914470A (en) * | 2010-07-22 | 2010-12-15 | 山东省科学院生物研究所 | Acinetobacter calcoaceticus and culture method and application thereof |
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2011
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899404A (en) * | 2010-01-05 | 2010-12-01 | 广东省微生物研究所 | Acinetobacter calcoaceticus and application thereof in degrading decabromodiphenyl ether |
| CN101914470A (en) * | 2010-07-22 | 2010-12-15 | 山东省科学院生物研究所 | Acinetobacter calcoaceticus and culture method and application thereof |
Non-Patent Citations (1)
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102676418A (en) * | 2011-10-28 | 2012-09-19 | 中国科学院烟台海岸带研究所 | Application of acinetobacter calcoaceticus capable of degrading petroleum |
| CN102851236A (en) * | 2012-08-01 | 2013-01-02 | 上海交通大学 | Acinetobacter and construction method and application thereof |
| CN103013851A (en) * | 2012-10-08 | 2013-04-03 | 上海交通大学 | Complex microbial inoculant and applications thereof |
| CN106754473A (en) * | 2016-11-24 | 2017-05-31 | 中国科学院烟台海岸带研究所 | One planting sand good fortune bacillus and the application in open maritime environment pollution thing is repaired |
| CN107058156A (en) * | 2016-12-27 | 2017-08-18 | 北京市农林科学院 | Tetracycline antibiotics degradation bacteria strains, the microbial inoculum containing the bacterial strain and its application |
| CN107058156B (en) * | 2016-12-27 | 2020-01-31 | 北京市农林科学院 | Tetracycline antibiotic degrading strain, microbial inoculum containing strain and application thereof |
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